This R21 application is submitted in response to an RFA (#AT-07-005) entitled "Mechanisms of Immune Modulation." The primary objective of our proposed studies is to understand the molecular mechanism of action of an important complementary medicine, gambogic acid (GA) that has a long history of medicinal use. GA is a polyprenylated xanthone, with an ?, ?-unsaturated carbonyl group, isolated from gamboges, the resin from Garcinia morella and Garcinia hanburyi. Many botanicals with such a reactive carbon center are known to possess interesting pharmacological properties. In this regard, GA, which possesses anti-tumor activity, is thought to possess anti-inflammatory activities that have so far remained untested. Due to the presence of the ?,?-unsaturated carbonyl group, GA is a potent Michael acceptor that can covalently interact with free thiol groups in proteins, including transcription factors. Our preliminary results demonstrate an inhibitory effect of GA on the bacterial lipopolysaccharide (LPS)-induced activation of nuclear factor-?B (NF-?B). NF-?B is a major transcription factor responsible for inflammation-driven disease progression. We will extend these studies to elucidate the underlying molecular mechanism of immune modulation of inflammation by GA with a specific focus on the transcription of two NF-?B target pro-inflammatory genes, cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)-?. The specific hypothesis to be tested is that GA down-regulates the transcription of COX-2 and TNF? via the formation of covalent Michael adducts with protein components of the NF-?B cascade. The hypothesis will be tested using LPS-stimulated murine bone marrow-derived macrophages and validated in an in-vivo inflammation model, in C57/BL6 mice, in two Specific Aims: 1) To examine the effect of GA on the transcriptional regulation of COX-2 and TNF?, and 2) To elucidate the molecular mechanism of attenuation of NF-?B activation and function by GA. Studies will be focused on attenuation of inflammation by GA via the transcriptional regulation of COX-2 and TNF? and will involve the use of luciferase-based reporter and chromatin immunoprecipitation assays. Furthermore, the interaction of GA with protein components of the NF-?B pathway will be assessed by using biotinylated-GA in mass spectrometric and biochemical functional studies. Our long-term objective is to enhance the understanding of the molecular mechanism of action of botanicals used in complementary and alternative medicine for their role in immune regulation of inflammation. PUBLIC HEALTH REVELANCE: Gamboges, the resin from Garcinia morella and G. hanburyi that belong to the family Clusiaceae (Guttiferae), which are native to Asia, Australia, Southern Africa, and Polynesia, are rich in a polyprenylated xanthone, gambogic acid (GA). There is a long history of medicinal use of Garcinia extracts against many ailments. Interestingly, the anti-tumor activity of GA has been well demonstrated and is thought to arise partly due to the anti-inflammatory activity associated with GA. However, the anti-inflammatory activity of GA has not been tested. We propose to study the molecular basis of the down-regulation of NF-?B-dependent expression of pro-inflammatory genes by GA in in vitro and in vivo models of inflammation.